CN108690875A - The micro-array chip and application method with bar code of ospc gene and hereditary change are infected for screening - Google Patents

Abstract

The present invention is that the micro-array chip and application method with bar code of ospc gene and hereditary change are infected for screening, describes the detection method for detecting the gene mutation of DNA and/or RNA sample and quantity change in infectious microorganism in sample or sample.In various embodiments, the method includes carrying out multiplex polymerase chain re-action (PCR) with the primer simultaneously containing bar code and special target sequence oligonucleotides.Then PCR product the oligonucleotide hybridization special with bar code and is printed on the solid matrix of micro-array chip by the fluorochrome label on universal primer.This method can be used for a large amount of infectious microorganisms of screening or gene mutation related with disease, and gene examines the change of shellfish number or gene expression dose, including cancer and genetic disease.The bar code specific oligonucleotide acid being printed on after being serially diluted on micro-array chip can be used for quantifying for PCR product, and sxemiquantitative is carried out to target sequence present in sample.Component of the present invention, device and method are to detect and quantitative a variety of target sequences, including gene mutation provides rapid screening detection.

Description

For screening infect ospc gene and hereditary change micro-array chip with bar code and Application method

Technical field

Generally speaking the present invention relates to molecular biology, microbiology and oncologies.The various pieces packet of the present invention Various components, the device and method for detecting the nucleotide containing DNA and RNA are included, are the diagnosis of communicable disease and cancer Qualitative and quantitative information is provided with processing.Further, the part of the invention includes using to contain bar code and target sequence simultaneously The primer progress multiplex polymerase chain re-action (PCR) of specific oligonucleotide acid is simultaneously mutual with bar code with being printed on micro-array chip The oligonucleotides of benefit analyzes PCR product.Various components, facility and method of the present invention are the inspection of a variety of target sequences Survey and quantitatively provide rapid assay methods, including gene mutation.

Background technology

Following background technology is used to help reader and understands the present invention, and is not construed as the prior art.

Genome is the full gene or inhereditary material of biology.Biological required all information are made of and maintained coding Nucleotide forms.Term " nucleotide " is well known in the art." nucleotide " described here refer generally to DNA, RNA molecule or it Derivative, including base.Base includes, for example, (such as adenine " A ", guanine " G ", thymus gland purine " T " or born of the same parents are phonetic by DNA Pyridine " C ") or RNA (a such as A, mono- uracil " U " of a G or C) the middle natural generation occurred or derivative purine or pyrimidine Base.Term " nucleotide " includes term " oligonucleotides " and " polynucleotide ", they are all the subgenus of term " nucleotide ". Term " oligonucleotides " is molecule of the length between 3 to 100 bases.Term " polynucleotide " refer to length at least more than The individual molecule of 100 bases.

Generally speaking these definition refer to a single chain molecule, but can also include and single chain molecule in a particular embodiment Local, substantially or completely complementation an other chain.Therefore nucleotide also includes duplex molecule or three chain molecules, and it includes spies The one or more complementary strands or " complement ", this particular sequence of sequencing row include molecule.

PCR (PCR) is that the skill of target (or template) DNA molecular is expanded by archaeal dna polymerase (1,2) Art.This technology is used for various uses in clinical labororatory and research laboratory, including molecular cloning, DNA sequencing, prominent Become the forensic identification of analysis, medical diagnosis on disease, recombinant protein synthesis and identification individual.This method relies on the heating and cooling of thermal cycle Repetitive cycling, it is such the result is that DNA passes through archaeal dna polymerase unwinding and duplication.A pair is referred to as the short dna few nucleosides of primer The single-stranded specific amplified for being used for targeting regions of acid.Primer sequence is complementary with part target sequence.PCR reacts generally in thermal cycle It is carried out in reaction solution in instrument.Reaction solution generally includes following ingredients:DNA templates containing region of DNA domain to be amplified;With The 3&apos of each chain of DNA target mark;Hold complementary primer;Heat-staple archaeal dna polymerase such as Taq DNA polymerase, Pfu DNA Polymerase or Vent archaeal dna polymerases;Nucleoside triphosphate (dNTPs);Optimize the slow of archaeal dna polymerase vigor and stability Fliud flushing, including potassium, magnesium or manganese ion.

By gel electrophoresis, or the fluorescent dye by being combined with DNA or by with fluorochrome label and target sequence Complementary and as the oligonucleotides of probe, PCR product can be detected and quantified.There are many based on PCR technologies to purpose at present Gene is detected, but still has many defects, is had the method for improvement to be supplied, is detected to the target product of amplification And the method for improvement expands target gene.

Invention content

On the one hand, a kind of multiplex polymerase chain re-action (PCR) of present invention offer and the micro-array chip with bar code Change of the system in combination for screening and detection infectious microorganism or screening disease-correlative gene mutation or gene expression dose, Method including cancer and genetic disease:This method includes:Carry out the first and second wheel PCR reactions;Wherein, in first round PCR The primer used includes the primer pair with following properties:Other than the target distinguished sequence tested, the first primer is in 5' End is containing there are one additional sequence of barcodes;And the second primer of primer pair contains universal primer sequence, as all primer pairs With identical sequence.

In some specific embodiments, carries out the second wheel PCR and react the primer used by unique bar code few nucleosides Sour primer and universal primer composition;Wherein, universal primer report molecular labeling, such as biotin, fluorescent dye, fluorescence nano One kind in grain, quantum dot or chemiluminescence dye.

In some specific embodiments, the report molecule can be it is any allow carry out PCR product detection Molecule, including biotin, fluorescent dye.When biotin is by use, the report being tagged on avidin or Streptavidin point The signal of son transmitting is used for PCR product detection.

In some specific embodiments, the signal of the report molecular emission can be molecule, send out fluorescence And chemiluminescence, or fluorescence or chemiluminescence are sent out by enzymatic reaction, or directly naked eyes are visible as gold particle.

In some specific embodiments, wherein in the bar code micro-array chip be that there are hundreds and thousands of a points Micro-array chip, each point containing unique bar code oligonucleotides for capture second wheel PCR in is synthesized by universal primer DNA chain.

In some specific embodiments, wherein each sequence of barcodes can be printed on more than one point (point repeated);Alternatively, for each sequence of barcodes, there is sequence identical with bar code but there are 1 or multiple nucleosides in centre The internal standard oligonucleotides of sour site mispairing is printed on as bar code on micro-array chip;Preferably, point and interior punctuate are repeated It is used to measure the specificity and repeatability of detection.

In some specific embodiments, wherein the bar code micro-array chip is printed on surface of solids conduct Chip, or on the surface of solids of multi-pore channel, such as 96 orifice plates.

In some specific embodiments, wherein system used in this method or method can be used for clinic Bedside (POC) diagnosis detection in analysis, food security and environmental monitoring.

In some specific embodiments, system used in this method or method is used for following detection:Cause to pass The microorganism to catch an illness is all microorganisms for causing communicable disease, with or without clinical symptoms.These infectiousness are micro- Part in biology can cause cancer.

In some specific embodiments, wherein system is used for screening and inspection used in this method or method Mutation in cls gene group DNA.

In some specific embodiments, system includes the detection being mutated in cancer.

In some specific embodiments, system can be used for the screening and detection of rna level in sample.At some In specific embodiment, RNA includes mRNA, tiny RNA and non-coding RNA.Rna level can be used for medical diagnosis on disease and disease The monitoring of progress.

On the other hand, the present invention provides a kind of reagent of detection target target sequence, which includes carrying out first and second Take turns the reagent of PCR reactions;Wherein, the primer used in first round PCR includes the primer pair with following properties:In addition to being tested Target distinguished sequence other than, the first primer is in 5'End is containing there are one additional sequence of barcodes;And the second primer of primer pair contains There is universal primer sequence, there is identical sequence as all primer pairs.

On the other hand, the present invention provides a kind of device for detecting pcr amplification reaction product, which includes carrying item The micro-array chip of code includes specifically being matched with the sequence of barcodes on the primer that is used in pcr amplification reaction in micro-array chip Another sequence of barcodes.It is that there are hundreds and thousands of in the bar code micro-array chip in some specific embodiments The micro-array chip of point, each point are closed for capturing in the second wheel PCR by universal primer containing unique bar code oligonucleotides At DNA chain.In some specific embodiments, wherein each sequence of barcodes can be printed on more than one point (point repeated);Alternatively, for each sequence of barcodes, there is sequence identical with bar code but centre there are one or more nucleosides The internal standard oligonucleotides of sour site mispairing is printed on as bar code on micro-array chip;Preferably, point and interior punctuate are repeated It is used to measure the specificity and repeatability of detection.

Description of the drawings

Fig. 1 is the amplification principle exploded view with multiplexed PCR amplification microorganism target sequence.

Fig. 2 is the amplification principle exploded view with target sequence in multiplexed PCR amplification virus.

Fig. 3 is the amplification principle exploded view with multiplexed PCR amplification genome mutation.

Fig. 4 is principle exploded views of the RNA as the detection of molecular labeling in cancer.

Fig. 5 is the testing principle exploded view using the micro-array chip with sequence of barcodes.

Fig. 6 is that the amplified reaction of the present invention and the micro-array chip with sequence of barcodes carry out target nucleotide and be detected Step and principle exploded view.

Fig. 7 is the key step flow chart for the array of point mutation or the ARMS analyses for targeting allele.

Fig. 8 is the cancer detection principle exploded view using rna level as biomarker.

It is described in detail

PCR method

PCR product is often used in sequencing analysis to determine that sequence and genome change, or makees target for molecular cloning The functional analysis of sequence.It has been changed for different measurement purpose PCR methods.It is the example of various PCR below:

Reverse transcription PCR:This method is used for the detection of RNA and quantifies.In this case, RNA molecule passes through reverse first Record enzyme is converted into complementary DNA (cDNA) molecule, which is referred to as reverse transcription.Reverse transcriptase is with RNA templates and to use RNA 3'The enzyme of short primer guidance the first chain cDNA synthesis of termini-complementary.The combination of reverse transcriptase and PCR is sometimes referred to as reverse transcription PCR (RT-PCR), allows RNA molecule to detect, quantitative or clone.Common reverse transcriptase includes fowl myeloblastomatosis The anomaly of malicious (AMV) reverse transcriptase, moloney murine leukemia virus (M-MuLV) reverse transcriptase and its modification.RNA in sample Detection can be used for diagnosing RNA virus, such as human immunodeficiency virus (HIV), influenza virus, A type and Hepatitis C Virus, Dengue fever virus etc..The quantitative analyzing RNA molecule of mRNA (mRNA) and some non-coding RNA molecules, for example, Microrna and Long non-coding RNA is used as biomarker in the cancer diagnosis of the cancer patient of accurate medicine and patient stratification sometimes.

Quantitative PCR (qPCR):Starting quantity of this method for quantitatively detecting DNA, cDNA or RNA in sample.Fluorescence contaminates Material, such as Sybr are green, and oligonucleotide probe green or containing fluorescence Eva is included in PCR reactions.Amplified production is measured in real time Quantity can determine in sample with the presence or absence of target nucleotide sequences and its copy number in the sample.Quantitative PCR sometimes by Referred to as real-time PCR.

Digital pcr (dPCR):DPCR is another PCR method for target sequence in quantitative sample.By template DNA point Son dilutes and is separated into multiple compartments or fine droplet so that some drops are contained only there are one the target sequence copied, and other Drop can be free of any target sequence.PCR reactions are carried out in each drop.It, will be to target sequence at the end of PCR reacts In the presence of (positive amplification signal) or there is no (negative, no amplified signal) to be counted, to be copied to target table sequence in sample Shellfish number provides one and directly counts.

Multiplex PCR:Include multiple primer pairs in single PCR reactions, leads to the amplification of a variety of target sequences like that.This Method allows to expand and analyze several genes or target in single reaction.The annealing point of each primer pair must be optimized to Obtain best result.In order to analyze PCR product by gel electrophoresis, the size of amplicon, which will be different, to be come to one It is distinguished on a glue.Multiplex PCR is also used for the library construction of next-generation sequencing.

Nest-type PRC:Two groups of special primers of target sequence are used in two consecutive PCRs.It is a pair of in the first round reacts Primer is used to generate the product of target DNA, and the flank sequence of target sequence can also be included other than expected target sequence Row.Then product is used for the second wheel PCR with one group of primer, the Complementary binding sites of the primer are anti-in first round PCR In the product answered.Nest-type PRC is specific commonly used in the DNA cloning for increasing low abundance target sequence.

Allele specific pcr:Allele is one of a variety of alternative forms of mutually homogenic or identical genetic locus.Base The change of one or more nucleotide leads to different allele because in.Allele specific pcr can be used for detecting in gene The variation of mononucleotide and several nucleotide.Sometimes referred to as amplification tolerance abruptly-changing system (ARMS) PCR, a species-specific amplification is only The method of allele with one or several nucleotide diversities.If mutation or allele change are known, draw Object can be designed to its 3'End is contained around single nucleotide variations and matched base-pair buffers so that in stringent item Under part, matched allele is effectively expanded, and prevents the allele of amplification mispairing.Therefore, if existed in PCR reactions Given allele, then the primer pair of the allele-specific will generate product, but not generate every other replacement equipotential The product of gene.Allele specific pcr can also be carried out with modified oligonucleotides, the modified oligonucleotides with The combination of complementary nucleic acid sequences has higher thermal stability and specificity, but it cannot act as the primer of archaeal dna polymerase, because This can block amplification.For example, peptide nucleic acid (PNA) is clamped for PCR to analyze single base to mutation (3).

Asymmetric PCR:The method is used for a DNA chain of DNA amplification template, because only needing to expand in this case One in two complementary strands.Product can be used for being sequenced and/or hybridize.PCR is carried out as usual, in addition to only needing addition one in the reaction Kind primer.Alternatively, in the case of two kinds of primers are added in the reaction, the addition much less of one of them.

Modified PCR.The dNTP of modification can be integrated into DNA during PCR, play the work(of report in the detection Energy (4).Mark the principle of PCR product similar to nick translation and random labelling method with the PCR of the dNTP of modification.Pyrimidine The modification of 7 nucleobase major grooves of 5 and purine is led in the labeling process of target sequence polymerase-mediated DNA Often with there is better efficiency.Connect decorating molecule (such as biotin, fluorescent molecular) and nucleotide linking arm flexibility with Length will have an impact dNTP substrate attributes.For example, biotin -4-dUTP, biotin -11-dUTP and biotin -16- DUTP represents the dUTP of the biotin labeling with different length linking arm.The dUTP of biotin labeling is integrated into PCR products On, to allow to detect PCR product with the report molecule of avidin or marked by streptavidin.

The PCR of primer with modification:In PCR reactions the primer that uses can be modified with increase its specificity and/ Or reduce its length.The modification for increasing primer specificity is in 5'End is conjugated (5) integration with minor groove binding and is incorporated to lock core Sour (6).

Isothermal amplification:Presently, there are a variety of isothermal amplification techniques.In the conventional method, most methods be from DNA cloning is generated in ssDNA templates.Noticeable exception is the amplification (NASBA) of the nucleic acid sequence side similar with some Method does not have the direct cloning RNA of reverse transcriptase step, this is by RNA viral diagnosis institute keen anticipation.Isothermal is developed The shortcomings that strand displacement polymeric enzyme reaction (ISDPR) overcomes PCR.Thermal cycle is not needed in ISDPR, in cost and simplicity side Face provides notable advantage.

Above-mentioned many round pcrs can be used for the detection of target sequence as shown in the present invention.

Micro-array chip

Micro-array chip is the device for the microcosmic spot set that its surface of solids has some biomaterials.Point sample is in array On biomaterial, such as DNA, protein, tissue etc., they are also referred to as DNA microarray, protein microarray and organize micro- Array etc..DNA microarray is known in the art, and for measuring a large amount of while expression gene level or to some gene The multiple regions of group carry out Genotyping (7-9).

Each DNA points contain several picomoles (10^-12Mole) identical DNA chain, referred to as probe or oligonucleotides, by with Hybridize in DNA the and cDNA molecules in sample.By detecting the target of fluorogen, silver or chemiluminescent labeling, usually detect Hybridize with quantitative probe-target mark, to determine the relative abundance of nucleic acids in samples sequence.The principle of DNA microarray, which is two, to be had Hybridization between the nucleic acid chains of complementary series, hydrogen bond can be formed and specificity is matched each other between complementary nucleotide base pair by having It is right., can be by fluorescence after washing removal non-specific binding sequence with suitable buffer solution, silver or chemiluminescent agent inspection Measure and be printed on the strong marriage chain of the probe or oligonucleotides specificity on array.Signal strength from spot with spot On the quantity of target that combines of probe it is related, the relative quantity of target sequence in sample is provided.

The probe used in DNA microarray can be used as spot printing on a solid surface, such as glass, plastics and micro- Pearl.Each spot is containing there are one identical probes.There are different holes on single slide, the surface of solids of the form of core piece or plate Cave quantity such as 24,48,96 orifice plates, hundreds and thousands of a spots are arranged in above with orderly row and column.Each spot is accurate Position and sequence are recorded in Computer Database, and for detecting the target sequence in sample.The present invention will use point sample Sequence bar code on the micro-array detects and quantifies the target nucleic acid sequence in sample.

The detection of special derivative biomolecule is typically used to the discriminating of infectious disease in infectious microorganism in sample Diagnosis, including by virus, bacterium and fungus-caused disease.These biomolecule include antigen, protein and genome (RNA And DNA molecular).The genome of organism, RNA (for RNA virus) or DNA (for DNA virus and every other microorganism) It can be detected by using the PCR of organism special primer.For the micro- life of infectiousness using DNA as its Genomic material Object, such as DNA virus and other most of microorganisms, including mycoplasma, Chlamydia, bacterium and fungi, genome can be direct Template as PCR analyses.Genomic DNA can be detached from sample by using the method based on liquid or solid matrix. Some of methods remove some inhibitor that may interfere with PCR reactions using specific reagent.

Because the genome of the primer reply detection microorganism used in PCR detections has high degree of specificity, PCR Detection has high degree of specificity for the confirmation of diagnosis.However a variety of in sample or unknown microorganism detection usually has Challenge.It therefore, can the method for multiple-microorganism will be helpful to clinical diagnosis in screening sample simultaneously.

Some microorganisms may experienced genome evolution, cause to generate resistance to some treatment processing, these processing It is effective to its wild type counterparts.In this case, it often requires many measure detects infectious microorganism, those micro- lifes The hypotype of object and specific resistant strain are oriented to for treatment and provide diagnostic message.Infectious microorganism, hypotype can be detected simultaneously Diagnosis required time and cost will be improved with the method for resistant strain.

The example that genome was made of DNA and can be used for the microorganism of PCR analyses is (but not limited to) aspergillus, yellow Aspergillus, aspergillus fumigatus, aspergillus nidulans, candida albicans, candida albicans, Cryptococcus neoformans, lattice Lu Shi cryptococcus, cryptococcus, herpesviral, second Hepatitis virus, herpes simplex virus 1-2, human cytomegalovirus, human cytomegalovirus, human cytomegalovirus, human cytomegalovirus disease Poison, mycoplasma pneumoniae, herpes virus hominis, JC viruses, papillomavirus 1-82, parvovirus B, false poxvirus, SV40 viruses, Vaccinia virus, varicellazoster virus and variola virus, some microorganisms may experienced genome evolution, cause to one A little treatment processing generate resistance, these processing are effective to its wild type counterparts.In this case, it often requires many measure Infectious microorganism is detected, the hypotype of those microorganisms and specific resistant strain are oriented to for treatment and provide diagnostic message.Energy Method that is enough while detecting infectious microorganism, hypotype and resistant strain will improve diagnosis required time and cost.

Genome be made of RNA and needed before PCR reverse transcriptase convert its RNA to cDNA virus include but It is not limited to astrovirus, Bu Niyawa viruses, california antigenic group viruses, Louis's encephalitis viruses, west nile virus, Japan Encephalitis viruses, eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, Murray Valley encephalitis viral, datum hole Agree refined virus, tick fever virus, hemorrhagic fever viruse, Coxsackie virus A 1-24, Coxsackie virus B1-6, dengue fever virus 1-4, Du Wen Haige (Duvenhage) viruses, Eastern equine encephalitis virus, Ebola virus, echo virus 1-24, enterovirus 1-71, intestines Road coronavirus, Hantaan virus, hepatitis A virus, Hepatitis C Virus, E viruses, human immunodeficiency virus (HIV) 1 and 2, Respiratory and enteric coronavirus, rotavirus, T- lymphocyte virus, influenza virus A, influenza virus B, Hu Ning (Junin) viruses are drawn Husky fever virus, measles virus, mumps virus, Norwalk virus, lymphocytic choriomeningitis virus, parainfluenza virus 1-4, poliovirus 1-3, hydrophobin, Respiratory Syncytial Virus(RSV), rhinovirus 1-113, Luo Xiou (Rocio) disease Poison, rubella virus, vesicular stomatitis virus, yellow fever virus, zika virus.

The embodiment of the present invention can be used for detection or a variety of diseases of screening or pathological condition, such as cancer.This can be passed through The cancer of method and the component assessment of invention includes cancer cell comprising comes from bladder, blood, bone, marrow, brain, mammary gland, knot Intestines, oesophagus, stomach and intestine, gum, head, kidney, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue or uterus it is thin Born of the same parents and cancer cell.

In addition, clear cancer has following histological type, although it is not limited to these:Tumour, it is pernicious;Cancer;Cancer, not Differentiation;Giant cell and spindle cell cancer;Small cell carcinoma;Papillary carcinoma;Squamous cell carcinoma;Lymphepithelioma;Basal cell Cancer;;Transitional cell carcinoma;Papillary transitional cell carcinoma;Gland cancer;Gastrinoma cholangiocarcinoma;Hepatocellular carcinoma;Joint hepatocellular carcinoma and courage Pipe cancer;Girder gland cancer;Adenoid cystic carcinoma;Adenomatous polyps gland cancer;Gland cancer, familial colon polyp;Solid carcinoma;Class cancer;It is pernicious swollen Tumor;Branch-alveolar adenocarcinoma;Papillary adenocarcinoma;Pigment cancer;Oncocytic carcinoma;Oncocytic adenoma;Basophilic granulocyte cancer;Thoroughly Clear cell adenocarcinoma;Granular cell carcinoma;Follicular adenocarcinoma;Mamillary and follicular adenocarcinoma;Non-packet film property hardens cancer;Adrenocortical carcinoma Endometrial carcinoma;The attached cancer of skin;Apocrine secretion gland cancer;Carcinoma of sebaceous glands;Adenocarcinoma of the uterine cervix;Mucoepidermoid carcinoma;Cystadenocarcinoma;Mamillary capsule Gland cancer;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Myxoadenocarcinoma;Signet ring cell cancer;Invasive ductal carcinoma;Cephaloma; Lobular carcinoma;Inflammatory carcinoma;Paget disease, mammary gland;Acinar cell carcinoma;Adenosquamous carcinoma;Gland cancer/squamous metaplasia;Thymoma ovary base Matter tumor, it is pernicious;Malignant tumour;Granular cell tumor, it is pernicious;Spongioblastoma;Sai Ertuolishi (sertoli) cell cancer;Leaching Bar cell tumour lipid cell tumour, it is pernicious;Chromaffionoma mammary gland ectoglia tumor, it is pernicious;Pheochromocytoma;Glomerulus Mesentery chromoma;Colourless property melanoma;Superficial spreading melanoma;Huge melanoma epithelioid cell melanoma;It is blue Mole sarcoma;Fibrosarcoma;Fibrous histiocytoma myxosarcoma;Embryonal-cell lipoma;Leiomyosarcoma rhabdomyosarcoma;Embryo's band Muscle tumor;Alveolar rhabdomyosarcoma;Stromal sarcoma mixed tumor;Endocervix (mullerian) mixed rumour;Kidney is female thin Born of the same parents' tumor;Hepatoblastoma;Carcinosarcoma mesenchymoma Robert Brenner tumour phyllodes tumor synovial sarcoma celiothelioma clone embryonal carcinoma;It is abnormal Tire tumor oophoroma choriocarcinoma;Middle kidney type angiosarcoma;Nemendothelioma, it is pernicious;Kaposi sarcoma;Hemangiopericytoma Lymphangioendothelial sarcoma osteosarcoma;Proximal end osteosarcoma;Chondrosarcoma chondrosarcoma mesenchymal chondrosarcoma;Giant cell tumor of bone;You Yin Sarcoma;Odontogenic tumor, it is pernicious;Ameloblastic otology sarcoma;Medulloblastoma ameloblastic fibrosarcoma;Pineal body Chordoma glioma ependymoma astrocytoma Primary Astrocytomas;Fibrous astrocytoma;At astrocyte Tumor;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal cerebellar sarcoma nerve is female thin Born of the same parents' tumor;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma neurofibrosarcoma;Neurinoma Granulocyte tumour, it is pernicious;Malignant lymphoma Hodgkin's disease;Hodgkin lymphoma;Accessory nerve pipe malignant lymphoma, primary lymphedema are thin Born of the same parents;Malignant lymphoma, maxicell, diffusivity;Malignant lymphoma, folliculus;Mycosis fungoides;Other specified non-Hodgkin's lymphs Tumor;Malignant histioctoysis increases;Huppert's disease;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukaemia;Lymph Sample leukaemia;Plasma cell leukemia;Erythroleukemia lymphosarcoma cell leukemia;Myelomatosis;Basophilic leukemia;It is thermophilic Acid leukaemia;Monocytic leukemia;Mast cell leukemia;Megakaryocytic leukemia;Myelosarcoma;With the white blood of hair cell Disease.In addition, the gene mutation and change of rna level are assessed as pre cancer, such as convert, anomaly sxtructure and hyperplasia.

The embodiment of the present invention can be used for detecting the gene mutation in cancer and genetic disease, examines shellfish art and changes or express water Flat change.The increase in demand of mutation analysis in the cancer and/or genetic disease of diagnosis sample.The base of various types of cancers Because of group feature it has been proved that with homolog origin, the cancer of histopathological diagnosis and clinical stage, their heredity changes Become and can be high diversity to the reaction of anticancer therapy.The success of it is now know that anticancer therapy is largely depended on In the ability of identification patient's subgroup, which can respond particular treatment reagent.

For example, the cancer patient with activated mutant will be from EGFR antagonisms in EGF-R ELISA (EGFR) gene Agent Gefitinib (10), Tarceva (11), Yi Luo is for benefited in the treatment of Buddhist nun (12) and Afatinib (13).Activate ALK bases Because (14) or ROS1 (15) genes and the cancer of genetic recombination can use ALK and ROS1 antagonist gram azoles to replace Buddhist nun (16), color is auspicious to be replaced Buddhist nun and Ai Le treat for Buddhist nun (17,18).Cancer with v-Raf murine sarcoma virus oncogene homologue B1 (BRAF) activated mutant BRAF inhibitor Wei Luofeini treatments (19) can be used;And poly- (ADP- ribose) polymerase (PARP) inhibitor Aura pa can be used Buddhist nun (20) treats the cancer of BRCA1 or BCRA2 gene mutations.Therefore, the mutation analysis of various cancer related genes is for treatment Cancer patient is provided needed for accurate pharmaceutical.

However, the different mutation of identical cancer related gene may have different influences to therapeutic response.For EGFR Gene, most common mutation are the L858R point mutation of the in-frame deletion and exon 21 of exons 19.In the lung cancer observed These mutation account for about 85% (21,22) together in EGFR mutation.With these mutation tumour for sieve replace Buddhist nun, Gefitinib and Yi Nuo is sensitive for the treatment height of Buddhist nun.The tumour of other more uncommon mutation, for example, on exon 18 G719 mutation and The mutation of L861Q on exon 21 can also use EGFR antagonist for treating (10).However, with being inserted into extron 20 frame Tumour, account for about the 4%-10% of all EGFR mutation, (23) usually do not reacted to the treatment of EGFR antagonists. T790M mutation in EGFR gene are usually detected in about 50% secondary drug resistance patient, these patients are initially to Lip river in distress For Buddhist nun, Gefitinib or nicotine have reaction but are changed into drug resistance or resistant (24) in rear extended meeting.Fortunately, have The cancer of EGFR T790M mutation can use third generation EGFR antagonist Moses to be treated for Buddhist nun (25).Therefore, these clinics are detected Mutation in sample is necessary for the accurate medication in treatment of cancer.

In one embodiment of cancer mutation analysis, it is known that many cancers usually have prominent in more than one gene Become.Mutation has been displayed to anticancer therapy with effect altogether caused by gene in other cancers.It was discovered by researchers that KRAS bases Because mutation or the dual-gene mutation of KRAS and TP53 cancer it is sensitive to docetaxel Jia Xiluomoni combination therapies, but KRAS and The cancer of the bis- mutation of STK11 is resistant to this combination therapy (26).Therefore, cancer diagnosis and accurate treatment will need simultaneously Detect the mutation in multiple regions (codon) and multiple genes.

In order to determine the mutation of cancer, the usual cancer cell present in the tumour or body fluid (such as blood, urine, phlegm etc.) Middle separation genomic DNA.It is being surgically removed or by biopsy samples obtain tumor tissues be usually used to un-mixing bases because Group DNA carries out mutant test.Some nearest researchs have shown that its DNA can be discharged into body fluid by cancer cell, Ke Yi (mutation, transposition are different for the change of detection tomour specific in extracellular DNA (also referred to as dissociative DNA, cfDNA) in the blood sample of cancer patient It often methylates and changes with copy number).CfDNA segments are usually present in body fluid from release in dead cell and with low concentration, including Blood plasma, serum, cerebrospinal fluid and urine (27).Cycle cfDNA can be detected in the blood plasma and serum of healthy individuals, but in urgency Property sports (28), pregnant (29) and increase its level under relevant pathological conditions with cell death and can increase, such as inflammation (30), myocardial infarction (31), burn (32) and graft rejection (33).

In addition, cycle cfDNA levels can also sharply increase (34) in the patient with various cancers.Some reports are aobvious Show, the blood plasma cfDNA detached from healthy individuals or benign disease patient is made of small fragment, these small fragments and nucleosome The incrementss (1-5x) of DNA (~150-200bp) (35,36) are corresponding, show that most of cycle cfDNA are thin from apoptosis Born of the same parents.Often detect the non-apoptotic DNA segment in blood plasma cfDNA (larger and smaller than withering in the sample obtained from cancer patient Dying property DNA fragmentation) quantity increase (36,37), show that the cfDNA in these patients from not only Apoptosis, is also wrapped Include necrosis, autophagy or mitosis obstacle.The test of the cycle cfDNA of cancer is also referred to as liquid biopsy.

It is expected with the barrier for overcoming tumor sample acquisition in conventional biopsy using the liquid biopsy test of cfDNA Hinder, because liquid biopsy is invasive smaller, cost is relatively low, is easy to carry out longitudinal dynamic evaluation from sample.This to be used for cancer Diagnosis detects for early-stage cancer screening and instructs accurately to cure for identifying the liquid biopsy that operable genome changes Interest is growing.Because recycling the half-life short (about 16 minutes) (38) of cfDNA, specific mutations is vertical in sample It can be used for monitoring therapeutic response, the appearance (39) of palindromia and treatment resistant mutants to Dynamic Variation Analysis.

In most cases, tumor tissues are made of cancer cell and normal cell, including stroma cell, such as fiber is female Cell, the lymphocyte and inflammatory cell of infiltration.Therefore, the genomic DNA detached from tumor tissues usually by normal DNA and The DNA of mutated tumor cell is formed.Often there are normal alleles and mutant allele for display for mutation analysis.Currently, The mutation analysis of cfDNA can use dPCR (40,41) or next-generation sequencings carry out (have group's cancer related gene (42, 43) cfDNA mutation).

Abrupt climatic change based on dPCR is limited to the special of the previously known cancer driving mutation being present in primary tumor Property hot spot, and the next-generation sequencing of cfDNA samples is limited to small set of genes (about 50 are reported in document) (42), 43).For facing Bed application, in order to which the early diagnosis of lung cancer and accurate treatment, the two are not in terms of the tumour-specific mutation for finding cfDNA Best.One of significant challenge using cfDNA detection tumour-specific mutation be can only be detached from blood plasma it is very limited CfDNA, this is not enough to use in the dPCR of the detection multiple hot spot mutations of cancer, or is not enough to be used for larger amount of cancer phase The genome analysis of correlation gene.

The embodiment of the present invention can detect in cfDNA multiple sites of one and/or multiple genes in unitary determination Gene mutation, solve the problems, such as to encounter in dPCR, i.e., can only in a double-colored channel measures (wild type and mutant) Detect a mutant.A large amount of cfDNA will be needed by detecting multiple mutation by dPCR, this is in most cases can not Capable.It also solves time and cost needed for next-generation sequencing analysis, and result is provided in a manner of time saving and high performance-price ratio. The method proposed in the present invention reduces the gap between dPCR and next-generation sequencing in the detection in Gene Mutation in liquid biopsy.

Specific implementation mode

Embodiment 1. is using DNA as the detection of the microorganism of genome

Fig. 1 pieces show the initial concentration of the DNA genomes detection for infectious microorganism.In sample in microarray core Two-wheeled PCR reactions are carried out before piece plug-in unit, which contains the bar code oligonucleotides of thousands of 15bp to 25bp. In first round PCR reacts, the special primer pair of microbial genome is used.It is micro- with being printed on for each primer pair The identical one section of sequence in part of sequence of barcodes on array is added to the 5&apos of a primer;It holds (such as forward primer), then One universal primer sequence is added on another primer (reaction primer).

Then PCR product is used for the second wheel PCR, reverse primer only there are one, what is used in being reacted with the first round is logical It is identical with reverse primer sequences.This general reverse primer is also in 5'End report molecular labeling, such as biotin or fluorescence dye Material.Second wheel PCR is also completed with the asymmetric PCR of the only universal primer of report molecular labeling.After second wheel PCR reactions, instead Mixture is answered to be dripped in detection reagent equipment, when reaction mixture moves through nitrocellulose filter, product is hybridized Onto bar code oligonucleotide probe.After chromatography removes other impurities, hybridization can be determined from the concentration of report molecule Target infectious microorganism sequence and microbial genome copy number relative quantity.When universal primer biotin labeling, Gold particle, the Streptavidin of fluorochrome label or avidin can be used for visualizing the PCR product hybridized with reagent strip.When When being marked using gold, hybridization can be with direct visualizztion.The approach can detect a variety of infectious microorganisms in sample.PCR each time It is optimised that the primer concentration of reaction, PCR reaction buffers and PCR cycle are based on this purpose.

For each sequence of barcodes, at least two sequences can be printed on micro-array chip:One with design item Code sequence perfect match, for capturing the DNA chain from the second wheel PCR labels reacted, the other is identical but in centre There is the difference of 1-5 base (mispairing) in position.The oligonucleotides of mispairing plays internal standard in specific hybridization and reduces background and intersection The effect of hybridization signal.

Examples of implementation 2. detect RNA virus with PCR bar code micro-array chips

General procedure, method and principle with it is described in embodiment 1 identical.In sample with various sides described in embodiment 1 Before method is analyzed, a reverse transcription is carried out RNA is transformed to cDNA.The primer used in reverse transcription can be few deoxidation chest Thuja acid-dT (12-mer to 20-mer or their mixture), the primer pond of random primer or target RNA sequence specific.Few- DT primers are used for the reverse transcription of poly-adenosine RNA, such as mRNA.Random primer is 6-mer oligonucleotides and is used for non-poly The reverse transcription of polyadenylation RNA.Specific primer sequences are used to detect the RNA virus of one group of determination.Reverse transcription and the reaction of first round PCR It can be carried out in one-step method.

Examples of implementation 3.The detection of mutant and mutation allele in genomic DNA

General program, method and principle are identical as described in embodiment 1, and one kind of first round PCR reaction petrochina is drawn Object contains sequence of barcodes, can be with allele in 3'Terminal specific matches, such as mutation allele and single nucleotide polymorphism, And the normal areas of the primer containing universal primer sequence and detected gene is matched.By the way that comprising some trims, equipotential is special The specificity of different primer can enhance, such as in the 3&apos of primer;It end, can be with the core of surveyed allele using a lock nucleotide Thuja acid matches.Sometimes, an additional mispairing is added to 3'End nearby increases the specificity of primer.This method can detect Various mutations in sample, the mutant being also used in detection cancer specimen, including primary tumor and liquid living tissue are cut Piece sample.

Embodiment 4.RNA is as molecular labeling cancer detection

The change of rna level in cell, such as mRNA, tiny RNA and long non-coding RNA are used for the biomarker of cancer. This purposes also uses in method of the present invention.In the method and principle and embodiment 1-2 of intracellular detection rna level It is identical.RNA is converted to cDNA by reverse transcription first.Sample passes through PCR and microarray chip analysis.It is strong based on fluorescence signal It spends, the relative populations of RNA molecule can be measured in sample.

In the case where lacking any element specifically disclosed herein, limitation, may be implemented illustrated and described herein Invention.Used terms and expressions method is used as the term of explanation and unrestricted, and is not intended in these terms and table Up to any equivalent for excluding shown and described feature or part thereof in the use of method, and it should be realized that various remodeling exist All it is feasible in the scope of the present invention.It is therefore to be understood that although specifically being disclosed by various embodiments and optional feature The present invention, but the modifications and variations of concept as described herein can use by those of ordinary skill in the art, and recognize It is fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.

It is described herein or record article, patent, patent application and every other document and can electronically obtain The content of information include herein by reference, just as each individual publication by specific and single in full to a certain extent Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents And all material and information are incorporated into the right in the application.

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Claims (10)

1. detecting system (BDLF) group of a kind of multiplex polymerase chain re-action (PCR) and the micro-array chip with sequence of barcodes It shares in screening and detection infectious microorganism or the mutation of screening disease related gene group, includes the side of cancer and genetic disease Method:This method includes:Carry out the first and second wheel PCR reactions;Wherein, the primer used in first round PCR includes with following The primer pair of characteristic:Other than the target distinguished sequence tested, the first primer is in 5'End is containing there are one additional bar code sequences Row;And the second primer of primer pair contains universal primer sequence, has identical sequence as all primer pairs.

2. according to the method described in claim 1, wherein, it is few by unique bar code to carry out the primer that the second wheel PCR reactions use Nucleotide primer and universal primer composition;Wherein, universal primer report molecular labeling, as biotin, fluorescent dye, fluorescence are received One kind in rice grain, quantum particle or chemiluminescence dye.

3. according to the method described in claim 2, wherein, the report molecule can any allow to carry out PCR product inspection The molecule of survey, including biotin, fluorescent dye.When biotin is by use, be tagged to the report on avidin or enzyme-linked Avidin The signal for accusing molecular emission is used for PCR product detection.

4. according to the method described in claim 2, wherein, the signal of the report molecular emission can be molecule, send out Fluorescence and chemiluminescence, or fluorescence or chemiluminescence are sent out by enzymatic reaction, or directly naked eyes are visible as gold particle.

5. according to the method described in claim 1, wherein, there are hundreds and thousands of a points in the bar code micro-array chip Micro-array chip, each point, which containing unique bar code oligonucleotides is used to capture second, takes turns in PCR and is synthesized by universal primer DNA chain.

6. according to the method described in claim 5, wherein, each sequence of barcodes can be printed on more than one point;Or Person has sequence identical with bar code but centre has in one or more nucleotide site mispairing for each sequence of barcodes Mark oligonucleotides is printed on as bar code on micro-array chip;Preferably, it repeats point and interior punctuate be used to measure detection Specificity and repeatability.

7. according to the method described in claim 1 or 6, wherein the bar code micro-array chip is printed on the surface of solids On the surface of solids as chip, or in multiple well, such as 96 orifice plates.

8. according to the method described in claim 1, wherein, system used in this method or method can be used for clinical point Bedside (POC) diagnosis detection in analysis, food security and environmental monitoring.

9. according to the method described in claim 1, wherein, system used in this method or method is used for following detection:Draw The microorganism for playing infectious disease is all microorganisms for causing communicable disease, with or without clinical symptoms.These are infected Part in property microorganism can cause cancer.

10. according to the method described in claim 1, wherein, system is used for screening and inspection used in this method or method The change of shellfish number or the change of base expression are examined in mutation in cls gene group DNA.